脑缺血再灌注损伤过程中一氧化氮合酶的表达

背景:一氧化氮合酶(nitric oxide synthase,NOS)是一氧化氮(nitric oxide,NO)合成的关键因素,由于NO在体内易与氧和血红蛋白等物质结合而迅速失活,不易准确定量测定。因此,测定NOS活性是深入研究NO在脑缺血再灌注损伤发病机制的重要环节。目的:研究脑内不同类型NOS在脑缺血再灌注损伤过程中的作用。设计:随机对照的动物实验。单位:青岛大学医学院附属医院脑血管病研究所。材料:实验于2005-05/12在山东省脑病防治重点实验室完成。选择成年健康雄性Wistar大鼠28只,清洁级,体质量220~260g,由山东大学实验动物中心提供。按随机数字表法分为假手术组和脑缺血组,假手术组4只,脑缺血组24只。脑缺血组又分为缺血1h再灌注6h,12h,1d,3d,7d,14d6个时间点,其中每个时间点4只。方法:应用线栓法经左侧颈外-内动脉插线建立大鼠大脑中动脉闭塞再灌注模型。应用免疫组织化学技术检测脑缺血再灌注后不同时间点脑内不同类型NOS的表达。主要观察指标:①甲苯胺蓝染色的两组神经细胞;②脑缺血组大鼠脑内神经元型NOS(neuronalNOS,nNOS)、内皮型NOS(endothelial NOS,eNOS)和诱导型NOS(inducible NOS,iNOS)在不同时间点的表达和分布。结果:①脑缺血组损伤区神经细胞出现核固缩、细胞碎片等,各时间点间细胞差异无显著性。②再灌注后6h脑内神经细胞即出现nNOS,eNOS和iNOS表达,随着再灌注时间的延长逐渐增强,脑组织神经元细胞不同类型NOS表达的区域一致,主要在皮质和纹状体区。脑内nNOS和iNOS于再灌注12h~7d保持较高的表达水平,而eNOS于再灌注6h~3d保持较高水平,持续时间短,升高和降低时间均早于nNOS和iNOS;但3种NOS均于再灌注1d到达表达高峰。3种NOS在皮质区和纹状体区的表达变化趋势基本一致。结论:脑缺血再灌注损伤后eNOS高表达时间较早,持续时间短,而nNOS和iNOS高表达时间稍迟,持续时间长。

Expression of nitric oxide synthase in the process of cerebral ischemia/reperfusion

BACKGROUND: Nitric oxide synthase(NOS) is the key factor for the synthesis of nitric oxide (NO) . Because NO combines with oxygen, hemoglobin and other substances in vivo easily and deactivates quickly, and it is not exactly determined, so determining the activity of NOS is the important link for further studying the pathogenesis of NO in cerebral ischemia/reperfusion (I/R)injury.OBJECTIVE: To study the effect of different types of NOS in the process of cerebral I/R injury.DESIGN: Randomized and controlled animal experiment.SETTING: Instituteof Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University.MATERIALS: This experiment was carried out in the Shandong Key Laboratory of Prevention and Treatment for Encephalopathy from May to December 2005. Twenty-eight adult healthy male Wistar rats, of clean grade, weighing from 220 to 260 g, were provided by the Experimental Animal Center of Shandong University. The involved rats were randomly divided into sham-operation group (n =4) and cerebral ischemia group (n =24). Six time points were set in cerebral ischemia group: ischemia 1 hour reperfusion 6 hours, 12 hours, 1 day, 3 days, 7 days and 14 days, 4 rats at each time point.METHODS: Rat models of middle cerebral artery occlusion/reperfusion were established by suture-occluded method through inserting a suture into the left internal-external carotid artery. The expressions of different types of NOS at different time points after cerebral I/R were detected by immunohistochemical technique.MAIN OUTCOME MEASURES: ①Toluidine blue-stained two groups of nerve cells; ② The expression and distribution of neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS(iNOS) at different time points.RESULTS: ①Karyopyknosis and cell debris appeared in the nerve cells of the injured region of cerebral ischemia group,and there were no significant differences of cells among different time points. ② Six hours after reperfusion, the expressions of nNOS, eNOS and iNOS were found in the neurons of brain tissue and increased with the elongation of time of reperfusion. The regions in which different types of NOS in neurons of brain tissue were expressed were cortical area and corpora striata. nNOS and iNOS were highly expressed within 12 hours to 7 days after reperfusion in the brain, and eNOS was highly expressed within a short time period, i.e. 6 hours to 3 days after reperfusion. eNOS expression increasing and decreasing occurred earlier than nNOS and iNOS. But the expressions of three kinds of NOS all reached peak on the first day after reperfusion. The changing tendencies of the expression of three kinds of NOS in the cortical area and corpora striata were the same basically.CONCLUSION: After cerebral I/R injury, the high expression of eNOS occurs early and lasts for a short time, while that of nNOS and iNOS occurs late and lasts for a long time.